Michael R. Garrett

IL-10 improves cardiac remodeling after myocardial infarction by stimulating M2 macrophage polarization and fibroblast activation

Inflammation resolution is important for scar formation following myocardial infarction (MI) and requires the coordinated actions of macrophages and fibroblasts. In this study, we hypothesized that exogenous interleukin-10 (IL-10), an anti-inflammatory cytokine, promotes post-MI repair through actions on these cardiac cell types. To test this hypothesis, C57BL/6J mice (male, 3- to 6-month old, nxa0=xa024/group) were treated with saline or IL-10 (50xa0μg/kg/day) by osmotic mini-pump infusion starting at day (d) 1 post-MI and sacrificed at d7 post-MI. IL-10 infusion doubled plasma IL-10 concentrations by d7 post-MI. Despite similar infarct areas and mortality rates, IL-10 treatment significantly decreased LV dilation (1.6-fold for end-systolic volume and 1.4-fold for end-diastolic volume) and improved ejection fraction 1.8-fold (both pxa0<xa00.05). IL-10 treatment attenuated inflammation at d7 post-MI, evidenced by decreased numbers of Mac-3-positive macrophages in the infarct (pxa0<xa00.05). LV macrophages isolated from d7 post-MI mice treated with IL-10 showed significantly elevated gene expression of M2 markers (Arg1, Ym1, and Tgfb1; all pxa0<xa00.05). We further performed RNA-seq analysis on post-MI cardiac macrophages and identified 410 significantly different genes (155 increased, 225 decreased by IL-10 treatment). By functional network analysis grouping, the majority of genes (133 out of 410) were part of the cellular assembly and repair functional group. Of these, hyaluronidase 3 (Hyal3) was the most important feature identified by p value. IL-10 treatment decreased Hyal3 by 28%, which reduced hyaluronan degradation and limited collagen deposition (all pxa0<xa00.05). In addition, in vivo IL-10 treatment increased fibroblast activation (proliferation, migration, and collagen production), an effect that was both directly and indirectly influenced by macrophage M2 polarization. Combined, our results indicate that in vivo infusion of IL-10 post-MI improves the LV microenvironment to dampen inflammation and facilitate cardiac wound healing.

The Dahl salt-sensitive rat is a spontaneous model of superimposed preeclampsia

The mechanisms of the pathogenesis of preeclampsia, a leading cause of maternal morbidity and death worldwide, are poorly understood in part due to a lack of spontaneous animal models of the disease. We hypothesized that the Dahl salt-sensitive (S) rat, a genetic model of hypertension and kidney disease, is a spontaneous model of superimposed preeclampsia. The Dahl S was compared with the Sprague-Dawley (SD) rat, a strain with a well-characterized normal pregnancy, and the spontaneously hypertensive rat (SHR), a genetic model of hypertension that does not experience a preeclamptic phenotype despite preexisting hypertension. Mean arterial pressure (MAP, measured via telemetry) was elevated in the Dahl S and SHR before pregnancy, but hypertension was exacerbated during pregnancy only in Dahl S. In contrast, SD and SHR exhibited significant reductions in MAP consistent with normal pregnancy. Dahl S rats exhibited a severe increase in urinary protein excretion, glomerulomegaly, increased placental hypoxia, increased plasma soluble fms-like tyrosine kinase-1 (sFlt-1), and increased placental production of tumor necrosis factor-α (TNF-α). The Dahl S did not exhibit the expected decrease in uterine artery resistance during late pregnancy in contrast to the SD and SHR. Dahl S pups and litter sizes were smaller than in the SD. The Dahl S phenotype is consistent with many of the characteristics observed in human superimposed preeclampsia, and we propose that the Dahl S should be considered further as a spontaneous model to improve our understanding of the pathogenesis of superimposed preeclampsia and to identify and test new therapeutic targets for its treatment.

Sildenafil Treatment Ameliorates the Maternal Syndrome of Preeclampsia and Rescues Fetal Growth in the Dahl Salt-Sensitive Rat.

Preeclampsia, a hypertensive disorder of pregnancy, is detrimental to both mother and fetus. There is currently no effective treatment, but sildenafil, a phosphodiesterase-5 inhibitor, has been proposed as a potential therapy to reduce blood pressure and improve uteroplacental perfusion in preeclamptic patients. We hypothesized that sildenafil would improve the maternal syndrome and fetal outcomes in the Dahl S rat model of superimposed preeclampsia. Dahl S rats were mated, and half received sildenafil (50 mg/kg per day, via food) from day 10 through day 20 of pregnancy. The untreated Dahl S rats had a significant rise in blood pressure and a 2-fold increase in urinary protein excretion from baseline to late pregnancy; however, sildenafil-treated Dahl S rats exhibited ≈40 mmu2009Hg drops in blood pressure with no rise in protein excretion. Sildenafil also increased creatinine clearance and reduced nephrinuria and glomerulomegaly. Sildenafil treatment reduced the uterine artery resistance index during late pregnancy in the Dahl S rat and improved fetal outcomes (survival, weight, and litter size). In addition, 19% of all pups were resorbed in untreated rats, with no incidence of resorptions observed in the treated group. Furthermore, tumor necrosis factor-&agr;, endothelin-1, and oxidative stress, which are characteristically increased in women with preeclampsia and in experimental models of the disease, were reduced in treated rats. These data suggest that sildenafil improves the maternal syndrome of preeclampsia and blood flow to the fetoplacental unit, providing preclinical evidence to support the hypothesis that phosphodiesterase type 5 inhibition may be an important therapeutic target for the treatment of preeclampsia.

A small-molecule inhibitor of TRPC5 ion channels suppresses progressive kidney disease in animal models

Gaining a foothold on kidney disease? The leading cause of kidney disease worldwide is known as focal segmental glomerulosclerosis (FSGS). FSGS is associated with loss of podocytes, an unusual cell type critical for the kidneys blood filtration function. Podocytes form interdigitating foot processes that wrap around capillaries and prevent leakage of plasma proteins into urine (proteinuria). Zhou et al. suppressed proteinuria by preventing podocyte loss in two different rat models of kidney disease, using a compound that selectively inhibits the TRPC5 ion channel (see the Perspective by Chung and Shaw). In short-term studies, this compound had no detectable side effects. Thus, TRPC5 inhibitors may merit exploration as a therapy for progressive kidney disease. Science, this issue p. 1332; see also p. 1256 A drug inhibits kidney disease in rat models by preventing loss of podocytes, cells critical for blood filtration. Progressive kidney diseases are often associated with scarring of the kidney’s filtration unit, a condition called focal segmental glomerulosclerosis (FSGS). This scarring is due to loss of podocytes, cells critical for glomerular filtration, and leads to proteinuria and kidney failure. Inherited forms of FSGS are caused by Rac1-activating mutations, and Rac1 induces TRPC5 ion channel activity and cytoskeletal remodeling in podocytes. Whether TRPC5 activity mediates FSGS onset and progression is unknown. We identified a small molecule, AC1903, that specifically blocks TRPC5 channel activity in glomeruli of proteinuric rats. Chronic administration of AC1903 suppressed severe proteinuria and prevented podocyte loss in a transgenic rat model of FSGS. AC1903 also provided therapeutic benefit in a rat model of hypertensive proteinuric kidney disease. These data indicate that TRPC5 activity drives disease and that TRPC5 inhibitors may be valuable for the treatment of progressive kidney diseases.

Na/K-ATPase Signaling Regulates Collagen Synthesis Through microRNA-29b-3p in Cardiac Fibroblasts

Chronic kidney disease (CKD) is accompanied by cardiac fibrosis, hypertrophy, and dysfunction, which are commonly referred to as uremic cardiomyopathy. Our previous studies found that Na/K-ATPase ligands or 5/6th partial nephrectomy (PNx) induces cardiac fibrosis in rats and mice. The current study used in vitro and in vivo models to explore novel roles for microRNA in this mechanism of cardiac fibrosis formation. To accomplish this, we performed microRNA profiling with RT-qPCR based arrays on cardiac tissue from rats subjected to marinobufagenin (MBG) infusion or PNx. The analysis showed that a series of fibrosis-related microRNAs were dysregulated. Among the dysregulated microRNAs, microRNA (miR)-29b-3p, which directly targets mRNA of collagen, was consistently reduced in both PNx and MBG-infused animals. In vitro experiments demonstrated that treatment of primary cultures of adult rat cardiac fibroblasts with Na/K-ATPase ligands induced significant increases in the fibrosis marker, collagen protein, and mRNA expression compared with controls, whereas miR-29b-3p expression decreased >50%. Transfection of miR-29b-3p mimics into cardiac fibroblasts inhibited cardiotonic steroids-induced collagen synthesis. Moreover, a specific Na/K-ATPase signaling antagonist, pNaKtide, prevented ouabain-induced increases in collagen synthesis and decreases in miR-29b-3p expression in these cells. In conclusion, these data are the first to indicate that signaling through Na/K-ATPase regulates miRNAs and specifically, miR-29b-3p expression both in vivo and in vitro. Additionally, these data indicate that miR-29b-3p expression plays an important role in the formation of cardiac fibrosis in CKD.

Characterization of the development of renal injury in Type-1 diabetic Dahl salt-sensitive rats

The present study compared the progression of renal injury in Sprague-Dawley (SD) and Dahl salt-sensitive (SS) treated with streptozotocin (STZ). The rats received an injection of STZ (50 mg/kg ip) and an insulin pellet (2 U/day sc) to maintain the blood glucose levels between 400 and 600 mg/dl. Twelve weeks later, arterial pressure (143 ± 6 vs. 107 ± 8 mmHg) and proteinuria (557 ± 85 vs. 81 ± 6 mg/day) were significantly elevated in STZ-SS rats compared with the values observed in STZ-SD rats, respectively. The kidneys from STZ-SS rats exhibited thickening of glomerular basement membrane, mesangial expansion, severe glomerulosclerosis, renal interstitial fibrosis, and occasional glomerular nodule formation. In additional studies, treatment with a therapeutic dose of insulin (4 U/day sc) attenuated the development of proteinuria (212 ± 32 mg/day) and renal injury independent of changes in arterial pressure in STZ-SS rats. Since STZ-SS rats developed severe renal injury, we characterized the time course of changes in renal hemodynamics during the progression of renal injury. Nine weeks after diabetes onset, there was a 42% increase in glomerular filtration rate in STZ-SS rats vs. time-control SS rats with reduced renal blood flow. These results indicate that SS rats treated with STZ develop hyperfiltration and progressive proteinuria and display renal histological lesions characteristic of those seen in patients with diabetic nephropathy. Overall, this model may be useful to study signaling pathways and mechanisms that play a role in the progression of diabetes-induced renal disease and the development of new therapies to slow the progression of diabetic nephropathy.

Isolation and high-throughput sequencing of two closely linked epistatic hypertension susceptibility loci with a panel of bicongenic strains.

Interactions or epistasis between genetic factors may contribute to missing heritability. While linkage analyses detect epistasis, defining the limits of the interacting segments poses a significant challenge especially when the interactions are between loci in close proximity. The goal of the present study was to isolate two such epistatic blood pressure (BP) loci on rat chromosome 5. A panel of S.LEW bicongenic strains along with the corresponding monocongenic strains was constructed. BP of each set comprising of one bicongenic and two corresponding monocongenic strains were determined along with the parental Salt-sensitive (S) strain. Epistasis was observed in one out of four sets of congenic strains, wherein systolic blood pressures (SBP) of the two monocongenic strains S.LEW(5)x6Bx9x5a and S.LEW(5)x6Bx9x5b were comparable to that of S, but the SBP of the bicongenic strain S.LEW(5)x6Bx9x5 (157 ± 4.3 mmHg) was significantly lower than that of S (196 ± 6.8 mmHg, P < 0.001). A two-way ANOVA indicated significant interactions between the LEW alleles at the two loci. The interacting loci were 2.02 Mb apart and located within genomic segments spanning 7.77 and 4.18 Mb containing 7,360 and 2,753 candidate variants, respectively. The current study demonstrates definitive evidence for epistasis and provides genetic tools for further dissection of the isolated epistatic BP loci.

Periodontal-induced chronic inflammation triggers macrophage secretion of Ccl12 to inhibit fibroblast-mediated cardiac wound healing

Chronic inflammatory diseases, such as periodontal disease, associate with adverse wound healing in response to myocardial infarction (MI). The goal of this study was to elucidate the molecular basis for impaired cardiac wound healing in the setting of periodontal-induced chronic inflammation. Causal network analysis of 168 inflammatory and extracellular matrix genes revealed that chronic inflammation induced by a subseptic dose of Porphyromonas gingivalis lipopolysaccharide (LPS) exacerbated infarct expression of the proinflammatory cytokine Ccl12. Ccl12 prevented initiation of the reparative response by prolonging inflammation and inhibiting fibroblast conversion to myofibroblasts, resulting in diminished scar formation. Macrophage secretion of Ccl12 directly impaired fibronectin and collagen deposition and indirectly stimulated collagen degradation through upregulation of matrix metalloproteinase-2. In post-MI patients, circulating LPS levels strongly associated with the Ccl12 homologue monocyte chemotactic protein 1 (MCP-1). Patients with LPS levels ≥ 1 endotoxin units (EU)/ml (subseptic endotoxemia) at the time of hospitalization had increased end diastolic and systolic dimensions compared with post-MI patients with < 1 EU/ml, indicating that low yet pathological concentrations of circulating LPS adversely impact post-MI left ventricle (LV) remodeling by increasing MCP-1. Our study provides the first evidence to our knowledge that chronic inflammation inhibits reparative fibroblast activation and generates an unfavorable cardiac-healing environment through Ccl12-dependent mechanisms.

Renoprotective effects of C-peptide in the Dahl salt-sensitive rat

Previous studies have demonstrated that renoprotective effects of C-peptide in experimental models of diabetes-induced renal disease may be mediated via lowering blood glucose. The present study examined the renoprotective effects of C-peptide in a model of nondiabetic renal disease, the Dahl salt-sensitive (SS/jr) rat. SS/jr rats were placed on a 2% NaCl diet for 2 wk (HS2, resulting in mild to moderate renal injury) or 4 wk (HS4, resulting in advanced renal injury) and then received either vehicle (veh) or C-peptide (Cpep) for additional 4 wk. Urine albumin (UAE) and protein (UPE) excretion rates were measured at baseline (i.e., before initiation of veh or Cpep treatment) and 4 wk later (i.e., at the time of death). Glomerular permeability, indexes of glomerulosclerosis and tubulointerstitial fibrosis, the presence of inflammatory cells, and protein expression of transforming growth factor-β (TGF-β) and podocin were measured at the time of death. In HS2 + veh rats, UAE and UPE increased by 74 and 92%, respectively, from baseline and the time of death. While HS2 + Cpep attenuated this increase in UAE and UPE, HS4 + Cpep had no effect on these parameters. Similarly, HS2 + Cpep reduced glomerular permeability, tubulointerstitial fibrosis, renal inflammation, TGF-β, and podocin protein expression, while HS4 + Cpep had no effect. These studies indicate that C-peptide is renoprotective in nondiabetic experimental models with mild to moderate renal injury.

Macrophage overexpression of matrix metalloproteinase-9 in aged mice improves diastolic physiology and cardiac wound healing following myocardial infarction

Matrix metalloproteinase (MMP)-9 increases in the myocardium with advanced age and after myocardial infarction (MI). Because young transgenic (TG) mice overexpressing human MMP-9 only in macrophages show better outcomes post-MI, whereas aged TG mice show a worse aging phenotype, we wanted to evaluate the effect of aging superimposed on MI to see if the detrimental effect of aging counteracted the benefits of macrophage MMP-9 overexpression. We used 17- to 28-mo-old male and female C57BL/6J wild-type (WT) and TG mice ( n = 10-21 mice/group) to evaluate the effects of aging superimposed on MI. Despite similar infarct areas and mortality rates at day 7 post-MI, aging TG mice showed improved diastolic properties and remodeling index compared with WT mice (both P < 0.05). Macrophage numbers were higher in TG than WT mice at days 0 and 7 post-MI, and the post-MI increase was due to elevated cluster of differentiation 18 protein levels (all P < 0.05). RNA sequencing analysis of cardiac macrophages isolated from day 7 post-MI infarcts identified 1,276 statistically different (all P < 0.05) genes (994 increased and 282 decreased in TG mice). Reduced expression of vascular endothelial growth factor A, platelet-derived growth factor subunit A, and transforming growth factor-β3, along with elevated expression of tissue inhibitor of MMP-4, in macrophages revealed mechanisms of indirect downstream effects on fibroblasts and neovascularization. While collagen accumulation was enhanced in TG mice compared with WT mice at days 0 and 7 post-MI ( P < 0.05 for both), the post-MI collagen cross-linking ratio was higher in WT mice ( P < 0.05), consistent with increased diastolic volumes. Vessel numbers [by Griffonia ( Bandeiraea) simplicifolia lectin I staining] were decreased in TG mice compared with WT mice at days 0 and 7 post-MI ( P < 0.05 for both). In conclusion, macrophage-derived MMP-9 improved post-MI cardiac wound healing through direct and indirect mechanisms to improve diastolic physiology and remodeling. NEW & NOTEWORTHY Aging mice with macrophage overexpression of matrix metalloproteinase-9 have increased macrophage numbers 7 days after myocardial infarction, resulting in improved diastolic physiology and left ventricular remodeling through effects on cardiac wound healing.